Optimal Hypoxia Regulates Human iPSC-Derived Liver Bud Differentiation through Intercellular TGFB Signaling.

Timely controlled oxygen (O2) delivery is crucial for the developing liver. However, the influence of O2 on intercellular communication during hepatogenesis is unclear. Using a human induced pluripotent stem cell-derived liver bud (hiPSC-LB) model, we found hypoxia induced with an O2-permeable plate promoted hepatic differentiation accompanied by TGFB1 and TGFB3 suppression. Conversely, extensive hypoxia generated with an O2-non-permeable plate elevated TGFBs and cholangiocyte marker expression. Single-cell RNA sequencing revealed that TGFB1 and TGFB3 are primarily expressed in the human liver mesenchyme and endothelium similar to in the hiPSC-LBs. Stromal cell-specific RNA interferences indicated the importance of TGFB signaling for hepatocytic differentiation in hiPSC-LB. Consistently, during mouse liver development, the Hif1a-mediated developmental hypoxic response is positively correlated with TGFB1 expression. These data provide insights into the mechanism that hypoxia-stimulated signals in mesenchyme and endothelium, likely through TGFB1, promote hepatoblast differentiation prior to fetal circulation establishment.

Evaluation of bioactivity of octacalcium phosphate using osteoblastic cell aggregates on a spheroid culture device.

Much attention has been paid to three-dimensional cell culture systems in the field of regenerative medicine, since three-dimensional cellular aggregates, or spheroids, are thought to better mimic the in vivo microenvironments compared to conventional monolayer cultured cells. Synthetic calcium phosphate (CaP) materials are widely used as bone substitute materials in orthopedic and dental surgeries. Here we have developed a technique for constructing a hybrid spheroid consisting of mesenchymal stem cells (MSCs) and synthetic CaP materials using a spheroid culture device. We found that the device is able to generate uniform-sized CaP/cell hybrid spheroids rapidly and easily. The results showed that the extent of osteoblastic differentiation from MSCs was different when cells were grown on octacalcium phosphate (OCP), hydroxyapatite (HA), or ß-tricalcium phosphate (ß-TCP). OCP showed the greatest ability to increase the alkaline phosphatase activity of the spheroid cells. The results suggest that the spheroids with incorporated OCP may be an effective implantable hybrid consisting of scaffold material and cells for bone regeneration. It is also possible that this CaP-cell spheroid system may be used as an in vitro method for assessing the osteogenic induction ability of CaP materials.